Brain metastases are a leading cause of mortality in cancer patients and there is currently no effective therapy that can prevent them. The search for therapies is limited by our poor understanding of the mechanisms of cancer cell brain invasion. We have recently discovered a novel mechanism of vascular plasticity that leads to the extravasation of emboli in the cerebral microcirculation. This process, which can clear any kind of material from the microvasculature, involves the engulfment of entire emboli by endothelial membrane projections and their subsequent translocation into the perivascular parenchyma. Our preliminary data shows that cancer cells undergo a similar process of microvascular engulfment. The goal of this proposal is to probe several potential roles of this mechanism in the metastatic process: a) cancer cells may co-opt this mechanism for the purpose of crossing the endothelial barrier and seeding the brain. This will be tested by using methods we have developed for transcranial two-photon imaging, confocal and electron microscopy. We will visualize at high spatial-temporal resolution the process of metastatic invasion in individual cerebral capillaries and determine if endothelial engulfment is required for cancer cell transvasation. b) the enveloping mechanism may help cancer cells remain insulated within microvessels promoting their latency. Using live and fixed tissue imaging and proliferation markers we will determine if cancer cells can remain dormant and viable for a long-term after microvessel engulfment. c) the engulfment process may serve as a surveillance mechanism by trapping and killing tumor cells within the vasculature. If the outcome of endothelial engulfment is death of malignant cells, we will monitor a variety of markers to characterize the death mechanism. Finally, we will examine several molecular pathways for their ability to modulate endothelial engulfment and will determine if this significantly impacts cancer cell transvasation, latency or survival. Together, these experiments will establish the importance of this mechanism of microvascular plasticity in the process of tumor invasion. Our results could suggest new targets for the prevention of brain metastasis. PUBLIC HEALTH RELEVANCE: Brain metastases are a leading cause of morbidity and mortality in cancer patients. Our limited understanding of the mechanism by which tumor cells enter the brain is hampering the search for preventive therapies. We have discovered a novel mechanism of microvascular plasticity that could have a critical role in the process of tumor cell brain invasion. This grant proposes to use animal models and imaging techniques that we have developed to test the importance of this mechanism in the process of tumor invasion and to probe the effect of its pharmacological inhibition on the progression of brain metastases.